The present invention relates to microbial genetic engineering practiced with the goal of developing new strains of microorganisms which are capable of dissimilating environmentally persistent chemical compounds.
Over the past several decades, numerous synthetic chemical compounds have been released into the environment in the form of insecticides, herbicides, propellants, lubricants, plasticizers, refrigerants, fire retardants and the like, with resultant accumulation of the compounds in the biosphere. In addition to commercially used synthetic compounds, large quantities of "waste" chemicals are formed during and after manufacture of synthetic compounds and have been stored by the chemical industry at waste dump sites. Production levels of synthetic organic chemicals have doubled every seven or eight years for the past three decades and annual production now exceeds 175,000,000 pounds per year.
Among the most widely used products of organic synthesis are the halogenated compounds. The introduction of halogen atoms (fluorine, chlorine, bromine, iodine) into organic molecules will frequently render them highly toxic to many insects, pests, weeds and pathogenic microorganisms. Such toxicity factors, coupled with relative ease of bulk synthesis, have made halogenated organic compounds, especially chlorinated aromatic compounds, highly desirable as herbicides and insecticides. Indeed, these compounds have been successfully used on a worldwide basis in concerted programs for the enhancement of agricultural productivity and elimination of insect vectors for transmission of disease.
The persistence of halogenated compounds in the environment for long periods after application was initially seen as a significant advantage. Most naturally-occurring microorganisms in soil and water simply do not possess the enzymatic capability for dissimilation of halogenated compounds (probably owing to the rarity of natural analogs of such compounds and the consequent absence of significant selective pressures favoring such capabilities). Because the compounds persisted in soil and water without being subjected to microbial degradation, repeated application of the herbicides and pesticides was not necessary.
Many halogenated aromatic compounds are lipophilic and not readily soluble in water. Herbicide and pesticide residues ingested by animals therefore tended to accumulate in fatty tissues, eventually making their way into the human food chain where they can have toxic, mutagenic and potentially carcinogenic effects. In addition to the gradual entry of such compounds into the food chain, humans have been subjected to hazard by the accidental release of large quantities of toxic compounds at manufacturing and storage facilities and along transportation routes. Pollution problems of tragic proportion in terms of human suffering and property damage have resulted from the well known episodes at Love Canal, NY, Seveso, Italy, and Yusho, Japan. Exposure of humans to high concentrations of persistent toxic compounds has also resulted from deliberate use of huge quantities of chlorinated aromatic hydrocarbons as defoliants in warfare. Finally, there are more than 3500 known waste dump sites in the United States alone where hazardous chemicals have been buried over the past decades and many of the dumps provide a potential or actual source of contamination of surface and ground waters near population centers.
The effective removal of environmentally persistent chemicals thus constitutes a problem of the highest order on a worldwide basis, with "target" compounds including: chlorinated dibenzo-p-dioxins (which are among the most highly toxic compounds known to man); 2,4,5-trichlorophenoxy acetic acid ("2,4,5-T", an Agent Orange constituent believed to include dioxins as manufacturing impurities and to generate dioxins upon combustion); chlorophenols; the mixed polychlorinated biphenyls ("PCB's"); lignin (in biomass conversion systems) and highly viscous recalcitrant components of oil (in secondary oil recovery systems). At present, there are few methods for dealing with contaminated industrial and municipal sludges or soils. It is possible to destroy most liquid toxic wastes completely with special high temperature/high oxygen incineration processing, but there is no easy disposal method available for contaminated soil or heavy sludges other than containment or burial.
Certain highly chlorinated compounds do appear to be metabolized by soil microorganisms by a mechanism known as co-oxidation. [See, e.g., Perry, Microbiol. Rev., 43, pp. 59-72 (1979).] Under co-oxidation conditions, the compounds are acted upon by individual members of the total microfloral population, each performing only a partial conversion of the substrate. The co-oxidation of a number of persistent chlorinated compounds such as 2,4,5-trichlorophenoxy acetic acid (2,4,5-T), 1,1-bis(p-chlorophenyl)-2,2,2'-trichloroethane (DDT) and [2-(2,4,5-trichlorophenoxy) propionic acid (Silvex) by mixed cultures is thus well known. Because individual microorganisms performing partial conversions rarely obtain energy from the conversion process, the entire conversion process is exceedingly slow and dependent upon the presence of other metabolizable carbon sources in the environment. Recent extensive studies of co-metabolizing mixed cultures capable of degrading, e.g., 2,4,5-T, reveal that co-oxidation processes can lead to chloride release, formation of phenolic products and/or cleavage of the armoatic ring, but that none of the microorganisms involved grow by using the herbicides as a sole carbon and/or energy source. See, e.g. Rosenberg, et al., J. Agric. Food. Chem.,28, pp. 705-709(1980) and Alexander, Science, 211, pp. 132-138 (1981).
While highly chlorinated compounds are recalcitrant to microbial attack except by co-oxidation, many simple halogenated compounds have been found to be susceptible to dissimilation by means of microbial degradation. Pure cultures have been isolated which are able to use mono-and di-chlorinated compounds such as 4-chlorobiphenyl, 3-chlorobenzoic acid, 2,4-dichlorophenoxyacetic acid and various fluoro- and chloro- acetates and propionates. See, e.g., Kamp, et al., at pp. 97-109. In "Plasmids of Medical, Environmental and Commercial Importance" Timmis, et al. (eds.) Elsevier/North-Holland Biomedical Press, Amsterdam (1979); Reineke, et al., J. Bacteriol., 142, pp. 467-473 (1980); Chatterjee, et al., J. Bacteriol., 146, pp. 639-646 (1981); Don, et al. J. Bacteriol., 145, pp. 681-686 (1981); Slater, et al., J. Gen. Microbiol., 114, pp. 125-136 (1979); and,
Kawasaki, et al., Agric. Biol. Chem., 45, pp. 1477-1481 (1981).
As in the case of petroleum degradative organisms (see, e.g., co-inventor Chakrabarty's U.S. Pat. No. 4,259,444), in many instances the degradative enzymes needed for microorganisms to dissimilate simple halogenated compounds are coded for by genes borne on DNA plasmids. While a single plasmid generally appears to encode only a single degradative pathway, plasmids often interact to greatly extend the number of xenobiotic compounds that can be degraded by a pure culture. An example of such interaction with regard to chlorinated aromatic compounds is found in the case of Pseudomonas species B-13 [see, Hartman, et al., Appl. Environ. Microbiol., 37, pp. 421-428 (1979)]. This strain, which could utilize 3-chlorobenzoate as a sole source of carbon, was subjected to introduction of the toluene oxygenase, "TOL", plasmid and it was thereafter possible to select variants of the strain which could also utilize 4-chlorobenzoic acid or 3,5-dichlorobenzoic acid as a carbon source.
There appear to be significant limitations, however, on the usefulness of painstaking plasmid manipulation and selection procedures. A case in point is the degradation of the chlorinated phenoxyacetic acids. Although several types of plasmids appear to have evolved for the degradation of 2,4-dichlorophenoxy acetic acid ("2,4-D"), deliberate and continued searches for the isolation of microorganisms capable of utilizing 2,4,5-trichlorophenoxy acetic acid as a sole carbon source have met with failure. See, e.g., Horvath, Bull. Environ. Contam. Toxicol., 5, p. 537 (1970); Rosenberg, et al., J. Agric. Food. Chem., 28, p. 297 (1980); Rosenberg, et al., J. Agric. Food. Chem., 28, pp. 705-709 (1980); Alexander, Science, 211, pp. 132-138 (1981); Don, et al., J. Bacteriol., 145 pp. 681-686 (1981).
Of significant interest to the background of the present invention are prevailing scientific theories of continuous culture of microorganisms and enrichment selection.
Microbial growth is termed balanced if there is a doubling of biomass accompanied by doubling of all other properties (e.g., protein, RNA, DNA) in order to maintain a constant chemical composition. Thus microbial growth rates can be studied by studying only one biochemical component. Bacteria growing in a rich medium within a growth flask typically show a classic sigmoidal growth curve, possessing four phases, i.e., lag, exponential, stationary, and death. The closed nature of a flask with its resulting characteristic growth curve is termed a "batch" culture, since the nutrients are not renewed and hence growth remains exponential for only a few generations. Although most laboratory cultures are grown as batch cultures, microbial species in nature virtually never reach batch conditions (unlimited growth). If the growth system becomes an open system, with nutrient renewal, as well as cell and expended medium removal, then it becomes possible to maintain a microbial population in an exponential state over a long period of time. When the average value of every individual cell property remains constant over time, a "steady state" of growth and division results. This steady state is analogous to the stationary phase of batch growth and can be generated in the laboratory via continuous culture.
Typically, a bacterial culture undergoing balanced growth mimics a first order autocatalytic chemical reaction, i.e., the rate of increase at any time (t) is proportional to the number (N) of bacteria. This relationship can be expressed mathematically as, ##EQU1## wherein: N.sub.o =the number of cells at time zero; t.sub.o =time zero; and .mu.=the growth rate constant.
A continuous culture is a flow system in which individual cells are suspended in a (nearly) constant volume, at or near a steady state of growth established by the continual addition of fresh growth medium, and the continual removal of part of the culture. In the simples mathematical form, ##STR1##
The rate of cell loss through overflow can be stated as: ##EQU2## wherein: N=number of cells
V.sub.O =culture volume (ml) PA1 [.omega.]=dilution rate PA1 f=flow rate (ml/hr)
Thus a stabilized steady culture will have .mu.=.omega.. Culture volumes have ranged from 10 ml to large scale industrial continuous culture fermentors, with dilution rates ranging from about 0.04 to 0.40 hour.sup.-1.
Since microbial growth mimics a first order chemical reaction, it is possible to derive a relationship for the nutrient concentration effect. Curves relating growth rate to nutrient concentration are typically hyperbolic and fit the equation: ##EQU3## wherein: .mu. is the specific growth rate at limiting nutrient concentration (C); .mu. max is the growth rate at saturating concentration of nutrient; and K.sub.s is an analogous constant to the Michaelis-Menten enzyme kinetic constant, being numerically equivalent to the substrate concentration supporting a growth rate equal to 1/2.mu.max. As an example, values of K.sub.s for glucose and tryptophan for Escherichia coli are 1.times.10.sup.-6 and 2.times.10.sup.-7 M, respectively, or 0.18 and 0.03 .mu.g/ml. These very low values are attributed to the high affinities characteristic of many bacterial permeases, which can be construed as an evolutionary adaptation to growth in extremely dilute solutions.
With continuous cultures, the growth rate equals the dilution rate in a stabilized system, thus ##EQU4## which states the fundamental relationship between substrate concentration (C) and dilution rate .omega..
Continuous culture systems can be operated either as chemostats or turbidostats. The former originated with Novick, et al., P.N.A.S., 36, pp. 708-719 1950) and Science,112, pp. 715-716 (1950) and with Monod, Ann. Inst. Pasteur., 79, pp. 390-410 (1950), whereas the latter was first reported on by Myers, et al., J. Gen. Physiol., 28, pp. 103-112 (1944). Every chemostat or turbidostat consists of four basic parts:
(1) A culture vessel (growth vessel or growth tube) in which cells are grown isolated from contamination by other organisms; PA0 (2) A nutrient supply system that delivers sterile nutrient medium at constant flow rate, and any gases needed; PA0 (3) A system for agitation of the culture, capable of rapid mixing of medium and gases required or produced and; PA0 (4) A system for drainage that removes fluid from the culture vessel at the same rate as fresh medium is supplied, and allows escape of gases.
In a chemostat the flow rate is set at a particular value and growth rate of the culture adjusts to this flow rate. In contrast, a turbidostat includes an optical-sensing device, e.g., photomultiplier tube, which measures culture absorbancy or density (either by transmitted or scattered light) in the vessel; the electrical signal from this device in turn regulates the flow rate. This results in the absorbancy of the culture determining the flow rate.
Continuous culture systems traditionally have offered two valuable features for microbial study. They provide a constant source of cells in an exponential phase of growth, and they also provide for cultures to be grown at extremely low substrate concentrations, similar to environmental concentrations. Growth at low substrate concentrations has classically been used in studies on regulation of synthesis and catabolism of the limiting substrate, as well as mutant production and ecological studies.